The members of the lipocalin protein family (Pervaiz, S., and Brew, K. (1987) FASEB J. 1, 209-214) are typically small, secreted proteins which are characterized by a range of different molecular-recognition properties: their ability to bind various, principally hydrophobic molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants), their binding to specific cell-surface receptors and their formation of macromolecular complexes. Although they have, in the past, been classified primarily as transport proteins, it is now clear that the lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and the synthesis of prostaglandins. The lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homoeostasis (reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14 and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).
The lipocalins share unusually low levels of overall sequence conservation, often with sequence identities of less than 20%. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded anti-parallel β-sheet closed back on itself to form a continuously hydrogen-bonded β-barrel. One end of the barrel is sterically blocked by the N-terminal peptide segment that runs across its bottom as well as three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and encompasses a target-binding site, which is formed by four peptide loops. It is this diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Flower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, or Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).
Human tear pre-albumin, now called tear lipocalin (TLPC), was originally described as a major protein of human tear fluid (approximately one third of the total protein content) but has recently also been identified in several other secretory tissues including prostate, nasal mucosa and tracheal mucosa. Homologous proteins have been found in rat, pig, dog and horse. Tear lipocalin is an unusual lipocalin member because of its high promiscuity for relative insoluble lipids and binding characteristics that differ from other members of this protein family (reviewed in Redl, B. (2000) Biochim. Biophys. Acta 1482, 241-248). A remarkable number of lipophilic compounds of different chemical classes such as fatty acids, fatty alcohols, phospholipids, glycolipids and cholesterol are endogenous ligands of this protein. Interestingly, in contrast to other lipocalins the strength of ligand (target) binding correlates with the length of the hydrocarbon tail both for alkyl amides and fatty acids. Thus, tear lipocalin binds most strongly the least soluble lipids (Glasgow, B. J. et al. (1995) Curr. Eye Res. 14, 363-372; Gasymov, O. K. et al. (1999) Biochim. Biophys. Acta 1433, 307-320).
The precise biological function of human tear lipocalin has not been fully elucidated so far and is still a matter of controversy. In tear fluid, it appears to be most important for the integrity of the tear film by removing lipids from the mucous surface of the eye to the liquid phase (reviewed in Gasymov, O. K. et al. (1999), supra). However, it displays additional activities in vitro that are very unusual among lipocalins, namely inhibition of cystein proteinases as well as non-specific endonuclease activity (van't Hof, W. et al. (1997) J. Biol. Chem. 272, 1837-1841; Yusifov, T. N. et al. (2000) Biochem. J. 347, 815-819). Recently, it has been demonstrated that tear lipocalin is able to bind several lipid peroxidation products in vitro resulting in the hypothesis that it might function as a physiological oxidative-stress-induced scavenger of potentially harmful lipophilic molecules (Lechner, M. et al. (2001) Biochem. J. 356, 129-135).
Proteins, which selectively bind to their corresponding targets by way of non-covalent interaction, play a crucial role as reagents in biotechnology, medicine, bioanalytics as well as in the biological and life sciences in general. Antibodies, i.e. immunoglobulins, are a prominent example of this class of proteins. Despite the manifold needs for such proteins in conjunction with recognition, binding and/or separation of ligands/targets, almost exclusively immunoglobulins are currently used. The application of other proteins with defined ligand-binding characteristics, for example the lectins, has remained restricted to special cases.
Rather recently, members of the lipocalin family have become subject of research concerning proteins having defined ligand-binding properties. The PCT publication WO 99/16873 discloses the class of so-called ANTICALINS®; i.e. polypeptides of the lipocalin family with mutated amino acid positions in the region of the four peptide loops, which are arranged at the end of the cylindrical β-barrel structure encompassing the binding pocket, and which correspond to those segments in the linear polypeptide sequence comprising the amino acid positions 28 to 45, 58 to 69, 86 to 99, and 114 to 129 of the bilin-binding protein of Pieris brassicae. The PCT publication WO 00/75308 discloses muteins of the bilin-binding protein, which specifically bind digoxigenin, whereas the International Patent Applications WO 03/029463 and WO 03/029471 relate to muteins of the human neutrophil gelatinase-associated lipocalin and apolipoprotein D, respectively. In order to further improve and fine tune ligand affinity, specificity as well as folding stability of a lipocalin variant various approaches using different members of the lipocalin family have been proposed (Skerra, A. (2001) Rev. Mol. Biotechnol. 74, 257-275; Schlehuber, S., and Skerra, A. (2002) Biophys. Chem. 96, 213-228), such as the replacement of additional amino acid residues.
However, for various applications it could also be advantageous to have more than one binding site per molecule available—either the natural binding pocket plus an engineered additional (protein)-binding site or two different engineered binding sites. For example, it could be considered to use lipocalin muteins as adapter or linker molecules which may be attached to a given binding partner via binding site I, whereas binding site II is used for screening/selection purposes or the like. One possibility to achieve this goal is the use of fusion proteins comprising two lipocalin muteins of same or different binding specificity, which are coupled to each other by a peptide linker. Such fusion proteins, also called “duocalins”, are described in WO 99/16873 and also by Schlehuber, S., and Skerra, A. (2001), Biol. Chem. 382, 1335-1342, for example.
Recently high-affinity histamine-binding proteins have been identified in the saliva of Rhipicephalus appendiculatus ticks (Paesen, G. C. et al. (1999) Mol. Cell 3, 661-671). These proteins sequester histamine at the wound site, outcompeting histamine receptors for the ligand in order to suppress inflammation during blood feeding. The crystal structure of these histamine-binding proteins has revealed a lipocalin fold novel in containing two binding sites for histamine having different binding affinities. The sites, one of which is a typical lipocalin binding site, are orthogonally arranged and highly rigid, forming an unusually polar internal surface that specifically complements the molecular properties of histamine. A related protein termed SHBP, which is secreted by a rodent- and cattle-feeding tick, binds both histamine and serotonin at the two different binding sites (Sangamnatdej, S. et al. (2002) Insect Mol. Biol. 11, 79-86). The high-affinity binding site lies perpendicular to the long axis of the β-barrel leading to distortions in the protein structure compared with other lipocalins. Thus, it appears as if such a binding site cannot be engineered in any given lipocalin. On the other hand, since the binding sites are rather buried in the core of the β-barrel there appear to be sterical limitations with regard to ligand size.
Thus, there remains a need for the generation of binding proteins that uses different binding sites and/or alternative lipocalin scaffolds, simply for the reason to have more options for practical realisation.